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- EMDB-42113: Tomograms of the apical end of Toxoplasma Gondii with Rhoptry Dis... -

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Entry
Database: EMDB / ID: EMD-42113
TitleTomograms of the apical end of Toxoplasma Gondii with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
Map dataToxoplasma gondii with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
Sample
  • Cell: Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
KeywordsRhoptry / Parasite / Conoid / CELL INVASION
Biological speciesToxoplasma gondii (eukaryote)
Methodelectron tomography / cryo EM
AuthorsMartinez M / Ben Chaabene R / Chang Y-W / Soldati-Favre D
Funding support United States, Switzerland, European Union, 4 items
OrganizationGrant numberCountry
David and Lucile Packard Foundation United States
Swiss National Science Foundation310030_185325 Switzerland
European Research Council (ERC)695596European Union
Burroughs Wellcome Fund1022785 United States
CitationJournal: PLoS Biol / Year: 2024
Title: Toxoplasma gondii rhoptry discharge factor 3 is essential for invasion and microtubule-associated vesicle biogenesis.
Authors: Rouaa Ben Chaabene / Matthew Martinez / Alessandro Bonavoglia / Bohumil Maco / Yi-Wei Chang / Gaëlle Lentini / Dominique Soldati-Favre /
Abstract: Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They ...Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They contain proteins and membranous materials injected directly into the host cells, participating in parasitophorous vacuole formation. Toxoplasma gondii tachyzoites harbor 8 to 12 rhoptries, 2 of which are docked to an apical vesicle (AV), a central element associated with a rhoptry secretory apparatus prior to injection into the host cell. This parasite is also equipped with 5 to 6 microtubule-associated vesicles, presumably serving as AV replenishment for iterative rhoptry discharge. Here, we characterized a rhoptry protein, rhoptry discharge factor 3 (RDF3), crucial for rhoptry discharge and invasion. RDF3 enters the secretory pathway, localizing near the AV and associated with the rhoptry bulb. Upon invasion, RDF3 dynamically delocalizes, suggesting a critical role at the time of rhoptry discharge. Cryo-electron tomography analysis of RDF3-depleted parasites reveals irregularity in microtubule-associated vesicles morphology, presumably impacting on their preparedness to function as an AV. Our findings suggest that RDF3 is priming the microtubule-associated vesicles for rhoptry discharge by a mechanism distinct from the rhoptry secretory apparatus contribution.
History
DepositionSep 25, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42113.png

Downloads & links

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Map

FileDownload / File: emd_42113.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationToxoplasma gondii with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.6 Å/pix.
x 800 pix.
= 8480. Å		10.6 Å/pix.
x 1440 pix.
= 15264.001 Å		10.6 Å/pix.
x 1023 pix.
= 10843.801 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.6 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1858.0 - 707.0
Average (Standard dev.)63.974789999999999 (±55.649006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-209209-401
Dimensions14401023800
Spacing10231440800
CellA: 10843.801 Å / B: 15264.001 Å / C: 8480.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Toxoplasma gondii with Rhoptry Discharge Factor 3 (RDF3)...

Fileemd_42113_additional_1.map
AnnotationToxoplasma gondii with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharg...

EntireName: Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
Components
  • Cell: Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down

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Supramolecule #1: Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharg...

SupramoleculeName: Toxoplasma Gondii tachyzoite apical complex with Rhoptry Discharge Factor 3 (RDF3) conditionally knocked down
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Toxoplasma gondii (eukaryote)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Details: DMEM supplemented with 5% fetal bovine serum and 2 mM glutamine
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 310 K / Instrument: LEICA EM GP / Details: Front blot for 4 seconds before plunging.
DetailsEgressed tachyzoites from a culture of confluent HFF cells. RDF3 knockdown induced by treatment with 50 nM rapamycin for 48 hours prior to egress.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Ted Pella, Inc / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
SoftwareName: SerialEM (ver. 3.8)
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.4 sec. / Average electron dose: 2.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsRaw frames were gain normalized in serialEM and motion corrected using the alignframes command in IMOD
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.11.5) / Number images used: 61

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